Figure 3.
At 6 h following T&HS lymphocyte count was reduced in blood and lung but static or increased in the lymphoid tissues. (A) Peripheral blood leucocyte count was determined manually on samples taken at intervals through the T&HS protocol. An initial leukocytosis at the onset of hemorrhagic shock was followed by leucopenia at 6 h. This returned to baseline by 24 h. C = 2.9 (2.6–3.1), 0 h = 5.3 (4.6–6.0), 2 h = 3.3 (2.5–4.0), 6 h = 1.7 (1.5–1.8), 24 h = 3.3 (3.0–3.5). Data are presented as mean (SEM) count × 106/ml and tested using Kruskal Walis and Dunn's multiple comparison post-test. (B) The lymphocyte population in blood, spleen and bone marrow was quantified, at intervals through the protocol, using flow cytometry. The lymphopenia demonstrated in blood at 6 h was not mirrored in the other lymphoid organ compartments. In spleen, lymphocyte count was static at 6 h but reduced by 24 h. In bone marrow, the lymphocyte count had increased by 6 h and remained above baseline at 24 h. Control(C) denotes a naïve subject of the same strain and age. C, 6 h, 24 h = Blood: 1.2 (0.03), 0.7 (0.11), 1.0 (0.18). Spleen: 56.7 (0.71), 57.5 (0.57), 36.3 (1.36). Bone marrow: 1.9 (0.43), 7.5 (0.77), 5.6 (0.93). Data are presented as mean (SEM) absolute cell count × 106/ml of blood/spleen/both hind limbs (n = 6–19). Data were tested using Kruskal Walis with Dunn's multiple comparisons test. All had a p-value < 0.01 and *denotes the positive comparisons. (C) The lymphocyte count in lung was examined at 6 h using flow cytometry. Lung was selected as a representative, non-lymphoid organ. By 6 h, the absolute count of lymphocytes in the lung tissue had reduced almost threefold C = 86 (84–87), 6 h = 31 (28–32), p < 0.01 (n = 6). Data are presented as mean (SEM) and tested with Mann Whitney U test, *denotes p < 0.05. (D) Evidence of widespread lymphocyte apoptosis was sought using a cell death assay on samples of blood, spleen, and bone marrow taken at 6 and 24 h following T&HS. Apoptosis was defined as Annexin V + 7AAD- and necrosis was defined as Annexin V+ 7AAD+. An increase in necrosis was observed in the spleen at 6 h. In all other organs and timepoints, there were no significant changes in apoptosis or cell death over the 24 h protocol. Blood: Apoptosis = 6 (6–9), 7 (7–10), 16 (15–16), p = 0.15. Necrosis = 2 (2–2), 7 (4–10), 1 (1–1), p = 0.08. Spleen: Apoptosis = 4 (4–7), 12 (10–13), 9 (9–9), p = 0.10, Necrosis = 5 (4–6), 29 (10–32), 6 (5–7), p = 0.02. Bone Marrow: Apoptosis = 4 (4–5), 12 (9–15), 12 (11–13), p = 0.10, Necrosis = 4 (3–5), 7 (6–8), 9 (8–9), p = 0.06. C denotes a naïve subject of the same strain and age. Data are presented as median (IQR) and tested with Kruskal Wallis and Dunn's comparison of all columns, *denotes p < 0.05.